The ultimate goal of this STTR proposal is to develop an innovative human tumor invasion assay for use as a screening tool in the identification of new anti-tumor therapeutics. Development of a model of early stage tumor invasion, the precursor condition to metastasis, coupled with a highly sensitive readout, is needed to identify new targets for drugs and/or agents that can be used to abrogate the invasion of primary H&N tumors and other types of carcinoma in humans. The NIKS(tm)-based human tumor model developed in the Allen-Hoffmann laboratory permits monitoring tumor cell growth within a normally developed, stratified squamous epithelium. Multiphoton imaging, an important tool for nondestructive investigation of living tissues, provides a highly sensitive readout for the detection of early changes in stroma underlying and/or surrounding tumors. We propose to expand upon our existing NIKS(tm)-NTM model by incorporating human tumor cells that possess a genetically engineered, invasive phenotype, and monitoring the behavior of these cells using multiphoton imaging. The following specific aims will be accomplished during Phase I: (1) Design and construct MT1-MMP expression vector, and demonstrate elevated MT1-MMP mRNA expression levels in transiently-transfected SCC13y cell monolayer cultures, (2) Demonstrate elevated protein expression levels and bioactivity in transiently-transfected SCC13y cell monolayer cultures, and (3) Develop stable, genetically-modified SCC13y cell clones, and evaluate bioactivity of these stable clones utilizing multiphoton microscopy. The unique combination of the novel stromal invasion model with a sophisticated imaging based readout will provide the pharmaceutical industry with a biologically relevant, human cell based, high throughput screening tool to evaluate novel cytostatic agents, and/or gene therapy strategies.